When an enzyme associated with a lysosome which is one of intracellular organelles is genetically defective or mutated, substances to be degraded or transported are accumulated as a foreign substance inside or outside cells. A disease of inborn error in metabolism caused by such a phenomenon is known as a lysosomal disease. Examples of the lysosomal disease include Niemann-Pick disease and GM1 gangliosidosis.
Niemann-Pick disease type C (NPC) is one of diseases of congenital lysosomal diseases caused by abnormality of a membrane protein NPC1 molecule which governs transportation of lipids mainly including cholesterol in cells or a secretory protein NPC2 molecule co-existing with NPC1 in endosomes. In patient's cells, free cholesterol and lipids are accumulated in lysosomes. NPC is characterized by hepatomegaly, splenomegaly, and a nervous symptom. NPC is a rare intractable disease which is developed at an infant stage, causes splenohepatomegaly or a progressive nerve disorder, and leads patients to death at around 10 years old. Effective therapy for the present disease has not been established.
A cyclic oligosaccharide, cyclodextrin (CyDs), is a monomolecular host molecule having hydrophobic hollow cavities in the molecule. When a guest molecule is taken into the hollow cavities of CyDs, to form inclusion complexes, a physicochemical nature of the guest molecule varies variously. The supramolecular inclusion phenomenon of CyDs called a molecular capsule is effectively utilized in many fields. Particularly, in drug development, the phenomenon is widely applied to improvement in preparation properties and construction of the drug delivery system.
Recently, Liu et al. have reported that when 2-hydroxypropyl-β-cyclodextrin (HPBCD) is intravenously administered to Npc1 gene-defective (Npc1−/−) mice, this is effective in improving the medical state or prolonging survival, and when HPBCD is directly administered into the brain, the improving effect thereof is increased a few hundreds times, compared with systemic administration (Non-Patent Document 1). Based on the outcome of these fundamental researches, U.S. FDA specially approved humanistic use of HPBCD to NPC child patients (intravenous administration and intrathecal administration). Under such background, also in Japan, in Hospital Affiliated to Medical Department of Sage University, HPBCD injectables were prepared in the hospital, and treatment of NPC child patients was initiated. As a result of continuation for more than 1 year of intravenous instillation of HPBCD (2500 mg/kg per time, 1 to 3 times per week) to NPC child patients, the certain effect of reduction of splenohepatomegaly and improvement in a brain wave in child patients was obtained, but a nervous symptom has not been improved yet. Then, in addition to HPBCD, a glycolipid synthesis inhibitor, Miglustat (50 or 100 mg per time, two times per day), was used concurrently. Furthermore, in order to directly deliver HPBCD into the brain not through the blood brain barrier, intrathecal administration and intraventricular administration via the Ommaya reservoir (30 mg/kg, once per week) are performed, concurrently with intravenous administration of HPBCD. Since treatment with HPBCD is first in Japan and there is no precedent of high dose administration and long term administration, the treatment is continued while the effectiveness and the harmful events of the treatment are closely examined. However, there is also a of the side effect, and HPBCD has not been generalized in Japan yet.
Meanwhile, HPBCD is approved as an additive (solubilizer) of medicaments, but a renal disorder is apprehended. In addition, events such as a pulmonary disorder have also been reported, and in the case of high dose administration or long term administration, safety thereof has become a problem. Accordingly, safer therapeutic agents for NPC in place of HPBCD are desired.
GM1 gangliosidosis is one of Gaucher diseases caused by a mutation of lysosomal-β-glucosidase which is a glycohydrolase, and a mutation of lysosomal-β-galactosidase is the etiology. This is a disease in which by deficiency of beta galactosidase, a glycolipid such as GM1-ganglioside and asialo-GM1-ganglioside, which is a substrate thereof, is accumulated in the brain or internal organs (liver, spleen) and the like, or a mucopolysaccharide such as keratan sulfate or the like is accumulated in the bone. There are three types including the baby type (type 1) which is developed at an early babyhood stage and associated with wide central nervous system disorders including spastic paraplegia, and a cherry red spot of the eyeground, splenohepatomegaly and bone abnormality, the juvenile type (type 2) which is developed from an infant stage and in which a central nervous system disorder progresses, and further, the adult type (type 3) in which a symptom such as dysarthria is manifested from a school age stage and an extrapyramidal symptom is a main symptom.
For these diseases, enzyme replenishment therapy has been main therapy until now, and examples of the problem include a problem that an enzyme preparation hardly reaches a central nerve, and the therapeutic effect on a nervous system including the brain is not seen, and a problem that dripping treatment with an enzyme preparation at the high cost must be continued through life. Accordingly, new therapeutic agents for these lysosomal diseases are desired.
Induced pluripotent stem cells (iPS cells), which are artificially produced from human somatic cells, can be induced to undergo sustained, unlimited growth and exhibit multipotency (i.e., the ability to give rise to various cell types in vitro). Because of these features, iPS cells have potential applications as a source of cell therapy in clinical medicine. The process of iPS cell generation, known as reprogramming, is triggered by the expression of four transcription factors, Oct3/4, Klf4, and c-Myc, which are the same core factors underlying pluripotency in other pluripotent stem cells such as embryonic stem (ES) cells. Overexpression of the four factors was initially mediated by lentivirus and retrovirus vectors in human skin-derived fibroblasts. Although these gene expression systems are stable, they have two potential problems in that the genes encoding the four factors are integrated into the host genome and remain in the resultant iPS cells, and there is a risk of insertional mutagenesis, can facilitate tumorigenesis in vivo
The development of efficient and safe reprograming methods based on the Cre/loxP recombination system, adenovirus vector, piggyback transposons, microRNA, and protein has suffered from a low frequency of iPS cell colony generation, a need for repetitive induction, and retention of a short length of foreign DNA in the host genome. A recent study showed that episomal plasmid vectors, which rarely integrate into the host genome, can be used to generate iPS cells from blood cells; however, the efficiency was low (˜0.1%) and factors such as p53 knock-down and the transient expression of EBNA were required in addition to the four reprogramming factors.
Sendai virus (SeV) vector technology is analternative strategy developed to overcome the obstacles described above. SeV vectors are minus-strand RNA viruses that express a gene of interest without integration into the host genome and have been used to efficiently generate iPS cells from human skin-derived fibroblasts and blood cells (Non-patent documents 2 and 3). The frequency of iPS cell colony generation with SeV vectors is higher than that achieved with conventional methods using retrovirus and lentivirus vectors (0.1% versus 0.01%). However, the SeV remains inside the cells for more than one month, and thus the establishment of transgene-free iPS cells requires a long time. Recently, the temperature-sensitive SeV (Ts-SeV) system was developed to prevent uncontrolled iPS cell generation due to the sustained cytoplasmic replication of SeV (Non-patent document 4). Ts-SeVs are easily and immediately eliminated from iPS cells derived from cord blood cells and fibroblasts by a temperature upshift, but the efficiency of iPS cell generation with current Ts-SeV vectors is low than that with SeV.
Further, method using the SeV has been reported for producing iPS cells from peripheral blood monocytes. In the method, a SeV vector continuously expressing reprogramming genes Oct4, Sox2, Klf4, and c-Myc is used and the removal of reprogramming gene mounted viral vector from cells is performed by using siRNA (Patent Document 1).
Although skin fibroblasts are the most common cell type used for generating iPS cells, skin biopsies are invasive and are not ideal for children or patients with skin diseases or coagulopathy. Peripheral blood cells is a preferable source cell; however, Ts-SeV vectors have not been reported for generating iPS cells from peripheral blood cells, and prolonged retention of SeV in iPS cells remains a problem when non-temperature sensitive SeV vectors are used.
Numerous iPS cell lines derived from the somatic cells of patients harboring pathogenic mutations, using methods including SeV, were shown to phenocopy the disease. Therefore these cell lines represent a powerful tool not only for cell therapy, but also for biomedical research and drug development. Biomaterial samples obtained from patients with intractable diseases are indispensable for studying the molecular mechanism of diseases and developing new therapeutic agents. However, because the number of samples from such patients is usually limited, disease-derived iPS cells are expected to be useful as a replacement or supplemental source of biomaterials for cell therapy. As just described, iPS cells have been used as a cell source or a cell model of disease. However, its use is limited by inefficient production and the presence of the transgene in cells. Therefore, a method for more efficiently producing iPS cells without introducing genes therein and safe is desired.